Quest Journals Journal of Research in Pharmaceutical Science Volume 2 ~ Issue 3 (2014) pp: 01-13 ISSN(Online) : 2347-2995 www.questjournals.org *Corresponding Author: 1 Dr Bharti Bhandari 1 | Page 1 Department of Physiology, AIIMS, Jodhpur Research Paper Antisense Oligonucleotide: Basic Concept and its Therapeutic Application 1 Dr Bharti Bhandari, 2 Dr Deepti Chopra, 3 Dr Neeta Wardhan, 1 Department of Physiology, AIIMS, Jodhpur 2 Department of Pharmacology, HIMSR, JamiaHamdard 3 Department of Pharmacology, UCMS, Delhi Abstract:- Antisense oligonucleotides are synthetic genetic materials that interact with natural genetic material and modulate them in a systematic way. Antisense oligonucleotides as a form of molecular medicine to modulate gene function was first acknowledged in the late 1970s. This therapy involves blocking translation, thereby inhibiting protein formation. Recently, antisense technology has been resurrected and has generated considerable enthusiasm in the research. Antisense oligonucleotides have proven to be valuable in gene functionalization and target validation and also represent a novel therapeutic strategy for wide range of diseases such as genetic disorders, cancers, and infectious diseases. Thus, in the present review an attempt is made to help the apprentice understand the basic concept of the antisense technology and its therapeutic applications. Keywords:- Antisense oligonucleotide, antisense technology, cancer, genetic disorders, Infections I. INTRODUCTION An antisense oligonucleotide [ASO]refers to a short synthetic strand of deoxyribonucleotide analogue that hybridizes with the complementary mRNA via Watson–Crick base pairing.ThemRNA in RNA-DNA duplexis a substrate for cellular Ribonuclease H [RNase H],an enzyme that destroys the RNA. RNase H cleaves the RNA-DNA duplex region of the mRNA thus induce a blockade in thetransfer of genetic information from DNA to protein. [1] Antisense oligonucleotides have been used to modify the expression of specific genes. [2]They are not only usefulin the study of loss-of-gene function and target validation, but also act as a novel therapeutic strategy to treat any disease that is linked to dysregulated gene expression [Table-1].Antisense oligonucleotides can also manipulate alternative splicing, thus can be usedto modulate the ratio of different splice variants or correct splicing defects[3]. II. MECHANISM OF ACTION ASO is taken up by cellular endocytosis, hybridize with the target mRNA resulting in the formation of ASO-mRNA heteroduplex leading in majority of times to: either activation of RNAse H or sterichindrance of ribosomal subunit binding.Both these mechanisms result in selective degradation of bound mRNA and ultimately target protein knockout. RNase H-dependent oligonucleotides can induce the degradation of mRNA when targeted to any region of the mRNA. However, the steric-blocker oligonucleotides physically avert the progression of splicing only when targeted to the 5‘or AUG initiation codon region. [4] Other mechanisms by which ASO can act is by entering the nucleus directly and altering maturation of mRNA,splicing activation, 5'-cap formation inhibition, arrest of translation and double strand RNAse activation. [5] III. OLIGONUCLEOTIDE ALTERATIONS Oligonucleotides with natural phosphodiester bonds have short stability and are highly susceptible to rapid degradation by intracellular endonucleases and exonucleases. Thus chemical modificationshave been developed to enhance nuclease resistance,cellular uptake, distribution, prolong tissuehalf-life, increase affinity and potency. [1] The modifications can be made to the nucleobases, sugar moiety [especially at the 2‘ position of the ribose] or phosphate backbone. [6] Oligonucleotides with modified sugar moieties and phosphate backbones are divided into three generations.
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3.1 First-generation ASOs- Phosphorothioate [Fig 1] First generation ASOs are those in which one of the non-bridging oxygen atoms in the phosphodiester bond is
replaced by a sulphur atom which introduces chirality at phosphorus The phosphorothioates are the most widely
studied oligonucleotides. The advantages of phosphorothioate oligonucleotide includes the relative ease of
synthesis and higher bioavailability by conferring higher resistance to the ASO against nuclease degradation,
and the. They are highly soluble and are also capable of activating RNase H. [4] However, the stability of a
phosphorothioate oligonucleotide has been shown to vary with each sequence and the cell line examined. [7]
Fig 1. Phosphorothioate DNA
The disadvantages of this modification are slight reduction in the affinity of the ASO for its mRNA target
because of decrease in the melting temperature of the ASO–mRNA heteroduplex approximately by 0.5 degree
C per nucleotide[8] and production of non-specific effects by interactions with cell surface and intracellular
proteins. [9, 10]
After a single application to tissue culture cells, the antisense effects of the phosphorothioates can be observed
for over 48 hours. [11]
Pharmacokinetics study of phosphorothioates in mice have demonstrated that following
intravenous or intraperitoneal administration, it is distributed in most of the tissues, is degraded mostly by
exonucleases and that up to 30% is excreted in urine in 24 hour and an additional 10% in 24-48 hours. [12]
Phosphorothioate oligonucleotide can be further modified by the addition of C-5 propyne pyrimidines to
increase their relative binding affinities and compensate for the decrease in melting temperature. Propyne-
modified oligonucleotide allow for a decrease in the length such that oligonucleotide as short as 11 bases can
have potent antisense effects. [13]
3.2 Second generation ASOs [Fig 2]
Second-generation ASOs with 2‘-O-alkyl modifications were developed to further enhance nuclease resistance
and increase binding affinity for target mRNA. 2‘-O-Methyl (2‘-O-Me) and 2‘-O-Methoxyethyl (2‘-O-MOE)
modifications ofPhosphorothioate -modified ASOs are the two most widely studied second-generation ASOs.
[14] Other substitutions that can be made at the ribose 2‘ position includes 2‘-fluoro, 2‘-O-propyl, and 2‘-O-
pentyl which can alter an oligonucleotide nuclease stability and binding properties. [15]These second
generation ASOs are less toxic than phosphorothioate oligonucleotide and have slightly greater affinity towards
their complementary RNAs.[15]
Fig 2. 2’-methoxyethyl RNA
Antisense effects of these second generation ASOs may be attributable to the steric block of translation.Methyl
and ethyl substitutions and 2‘ modified analogs were shown not to support RNase H-mediated cleavage of
target mRNA. [16]
However, the most desirable mechanism for antisense effect is the cleavage of target RNA by RNase H.
Gapmer technology is used to circumvent this shortcoming which consists of chimeric ASO with central ‗gap‘
region consisting of phosphorothioate oligonucleotide [sufficient to induce RNase H cleavage] is flanked on
both sides (5‘ and 3‘ directions) by nucleotide ‗wings‘ composed of 2‘-OMethyl or 2‘-Methoxyethyl modified
nucleotides. [6]
Antisense Oligonucleotide: Basic Concept and its Therapeutic Application
dioleoylphosphatidylethanolamine, when added into the liposomes allow the oligonucleotides to escape from
the endosomes.[39, 40] Liposomes or immunoliposomes have been demonstrated to increase the delivery of
synthetic antisense oligonucleotides into human myeloid and lymphoid leukaemia cells. [41]
Dendrimer are spherical and highly branched polymers with cationic polyamidoamine moieties
proficient of forming a covalent complex with the ASO. The dendrimer–ASO complex offers advantage over
the liposomal formulation by being stable and active even in the presence of serum. It enhances the delivery of
ASO into the cytosol and nucleus and also increases the retention time of ASO in the cells. [1] Starburst
polyamidoamine[PAMAM]dendrimers are a new type of cationic polymers with a molecular architecture
characterized by regular, dendric branching with radial symmetry and modest toxicity. [42,43]
ASO can also be conjugated to cell-penetrating peptides [CPP] that promote the cellular uptake of the
ASO. CPP are relatively short (9–30 amino acids)polycationic peptides rich in arginine and lysine, with net
positive charge. [44]Commonly used cell penetrating peptides include HIV-1 Tat protein, Transportan,
Antennapedia protein of Drosophila, synthetic Pep-1 peptide. [45] Some of the internalization mechanisms
proposed for the cellular uptake of CPPs include endocytosis and direct translocation or cell penetration. [46]
CPP-based systems appear to be very versatile and efficient.
Recently, various newer staragies have been used to improve delivery of ASO to its target like use of
monnosylated chitosan nanoparticles and conjugation with histidine rich peptide.[47, 48]
IV. BIOLOGICAL BARRIERS TO IN VIVO DELIVERY OF THE ASO A major biological barrier between oligonucleotide and its ultimate site of action (cytosol or nucleus)
is the rapid excretion via the kidney. Other major barriers include vascular endothelial wall, degradation by
serum and tissue nucleases, uptake by the phagocytes of the reticuloendothelial system, slow diffusion through
and binding in extracellular matrix and inefficient release from endosomes. [44]
V. TOXICOLOGY OF ASO In general, ASO drugs have shown to produce dose-dependent, transient and mild-to-moderate
toxicities manifested in rodents, primates and humans. Toxicity study of phosphorothioateoligodeoxynucleotide
and its analogues done in animals have shown to cause thrombocytopenia, dose-dependent elevation of liver
transaminases, reduction of the levels of alkaline phosphatase, albumin, and total protein. Splenomegaly,
PRO051 exon 51 of the dystrophin gene Duchenne's muscular dystrophy Phase 3
ATL1103 Growth Hormone receptor
(GHr)
Acromegaly, diabetic retinopathy Phase 1/2
Aganirsen Insulin Receptor Substrate-1
(IRS-1)
Corneal graft rejection,
Retinopathy of prematurity,
Neovascular Glaucoma, diabetic
retinopathy, Age related macular
degeneration
Phase 2/3
Trabedersen transforming growth factor-
beta 2 (TGF-β2
high grade glioma
malignant melanoma
pancreatic cancer
Phase 2
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